ABSTRACT Peripheral arterial disease (PAD) is highly prevalent and is increasingly recognized as a major contributor to the cardiovascular disease (CVD) and public health burden. Critical limb ischemia (CLI) is one of the most advanced PAD. To date, beside endovascular or surgical treatment, few therapeutic alternatives to restore the blood flow in ischemic tissues are available. Therefore, it would be of high significance to identify novel strategies to treat ischemic vascular injury in vivo. Transcription factor-EB (TFEB) is a crucial regulator of lysosomal biogenesis and autophagy. However, the functions of TFEB in vascular disease remain to be explored. The clinical detection and quantitation of nitric oxide (NO)-dependent fatty acid nitration products (nitroalkenes), has sparked the interest on novel anti-inflammatory lipids. Free and esterified nitro-fatty acid derivatives have been detected in human and animal plasma in the nanomolar range and shown to have profound implications in the prevention of diverse pathophysiological aspects of CVDs. Significant advances in metabolomics and lipidomics strategies identified conjugated linoleic acid (CLA) as the preferential substrate for fatty acid nitration in humans. Herein, we show that nitro-conjugated linoleic acid (nitro-CLA) formation readily translates into protective mechanisms in the vasculature. Our preliminary data indicate that nitro-CLA enhances autophagy through TFEB in endothelial cells (ECs). Endothelial TFEB significantly increases postischemic angiogenesis in vivo. Finally, nitro-CLA regulates autophagy and tube formation in a TFEB- dependent manner. Based on this evidence, the project will test the central hypothesis that enhancing the endogenous production of nitro-CLA protects against ischemic vascular injury by promoting TFEB- mediated autophagy leading to enhanced postischemic angiogenesis. Two Specific Aims are proposed. Aim 1: Establish that endothelial TFEB is essential for nitro-CLA-dependent protective autophagy and proangiogenic phenotype in vitro. This will be addressed by gain- and loss-of-function approaches in cultured primary ECs in the presence of nitro-CLA treatment. Aim 2: Establish that endogenous nitro-CLA protects from ischemic vascular injury in vivo through endothelial TFEB. An oral therapeutic strategy to promote endogenous nitro-CLA formation will be established. Unique EC-selective TFEB transgenic and knockout mice will be used to establish that TFEB function is required for nitro-CLA-promoting blood flow recovery in vivo. It is expected that with this proposal we will better define the essential role of TFEB in nitro-CLA-regulated EC proangiogenic phenotype and postischemic angiogenesis, and establish oral bioavailability of nitro-CLA as a novel therapeutic strategy against ischemic vascular injury. This mechanistic research will set a solid foundation for clinical utilization of nitro-CLA and lead to a major breakthrough for treating or/and preventing ischemic vascular disease by efficiently increasing postischemic angiogenesis.